Cell-free human immunodeficiency virus type I (HIV-1) virions are believed to affect intra- and inter-host transmission and in vivo pathogenesis. As such, the full evaluation of the molecular processes within HIV-1 virions may be critical towards the understanding of the natural history of HIV-1- induced disease. Reverse transcriptase catalyzes the synthesis of a proviral DNA intermediate, using the genomic viral RNAs as templates. Traditionally, it was assumed that this process occurred only in a target cell, which was infected by a retrovirus. However, recent data, from our laboratory and others, suggest that variable quantities of viral DNA are carried by HIV-1 virions. In addition, virion-associated HIV-1 DNA synthesis can occur in a simple reaction mixture within intact virions, without detergent permeabilization. This process, driven by deoxyribonucleotide triphosphates (dNTPs), may be entitled "natural endogenous reverse transcription" (NERT). As such, HIV-1 virions can be considered biochemically-active particles. Our laboratory now proposes to evaluate, in complementary specific aims, the molecular structure and mechanisms involved in producing intravirion HIV-1 DNA, and the relationship of NERT to the in vivo pathogenesis of HIV- 1. 1.) The process of NERT will be explored using polymerase chain reaction (PCR) techniques, direct radiolabelling of de novo synthesis of intravirion DNA, plus modified RNase protection assays and Southern blotting using strand specific probes, and certain modifying enzymes. Mutational analyses of specific viral proteins' effects (Envelope, Vpr, Vif) on NERT, and the interactions of specific inhibitors (i.e., viral protease inhibition) with intravirion DNA synthesis will also be evaluated. Molecular effects of critical polyamines, found in key human physiological fluids, on intravirion reverse transcription will be analyzed. These studies will interrelate with infectivity studies of HIV-1 virions harboring viral DNA, concentrating on non-proliferating primary human cells (i.e., quiescent T-lymphocytes and macrophages). Primary clinical viral isolates and molecular analyses of infectivity will be highlighted. 2.) NERT and intravirion reverse transcripts within differing clinical disease stages, and the effects on vertical and horizontal inter-host transmission of HIV-1, will be analyzed. These studies, evaluated in cross-sectional and longitudinal manners, will also address the levels of dNTPs required to induce NERT in various human physiological fluids, with and without other sexually transmitted diseases (STD). Intravirion DNA synthesis will be studied in blood plasma, seminal fluid, and cervical-vaginal secretions of HIV-1-infected-individuals, including individuals with reverse transcriptase-inhibitor resistant viral strains. Thus, these focused and interrelating experiments will allow precise analyses of the molecular structure and mechanistic relationships during NERT, and determine the effects of intravirion DNA synthesis on HIV-1 pathogenesis in vivo.